Estrogenic compounds and animal growth promoters



United States Patent 3,239,346 ESTRUGENIC COMPOUNDS AND ANIMAL GROWTH PROMOTERS Edward B. Hodge, Phil ll-I. Hidy, and Herbert L. Wehrmeister, Terre Haute, Ind., assignors to Commercial Solvents Corporation, a corporation of Maryland No Drawing. Filed Feb. 15, 1965, Ser. No. 432,827 26 Claims. (Cl. 992) The present invention relates to new compounds and an object of the present invention is to provide compounds which exhibit estrogenic activity or aid in increasing the rate of growth in meat-producing animals, e.g. cattle, lamb and swine.

A conventional formula for the compounds of the present invention is:

where A is -CH CH R is hydrogen or substituted or unsubstituted alkyl, e.g. lower alkyl such as methyl, ethyl, and hcxyl, etc., but hydrogen is preferred, R is hydrogen, substituted or unsubstituted alkyl, e.g. lower alkyl, or unsubstituted or substituted aryl, e.g. a monoring aromatic such as phenyl, tolyl and bromophenyl and R is hydrogen. Compounds having the above formula wherein A is -OH=CH; R is unsubstituted or substituted aryl, e.g. phenyl and bromophenyl; acyl, e.g. acetyl and valeryl; alkenyl, e.g. lower alkenyl such as allyl, butenyl and pentenyl; and aralkyl, e.g. benzyl; R is alkenyl, e.g. lower alkenyl such as allyl, 'butenyl and pentenyl; and R is alkyl, e.g. lower alkyl such as methyl, hexyl etc. and aryl, e.g. phenyl, as well as the nontoxic salts of the compounds such as the oxalates and sulfates are also contemplated by the present invention. There are two diastereoisomers of the compounds of the present invention.

The compounds can be administered to animals by any suitable method including oral and parenteral administrations. For example, the compound can be blended with ordinary feed containing nutritional values in an amount sufiicient to produce the desired rate of growth and thus be fed directly to the animals, or the compound can be suspended in a suitable injection suspension medium such as peanut oil and injected parenterally. The amount of compound fed to an animal, of course, varies depending upon the animal, desired rate of growth and the like.

When the new products are to be administered in feeds, an animal feed composition may be prepared containing the usual nutritionally-balanced quantities of carbohydrates, proteins, vitamins and minerals, together with the compound of the present invention. Some of these usual dietary elements are grains, such as ground grain and grain by-products; animal protein substances, such as those found in fish meal and meat scraps; vegetable protein-s like soybean oil meal or peanut oil meal; vitaminaceous materials, e.g. vitamin A and D mixtures; riboflavin supplements and other vitamin B complex members; and bone meal and limestone to provide minerals. A type of conventional feed material for use with cattle includes alfalfa hay and ground corn cobs together with supplementary vitaminaceous substances if desired.

Patented Mar. 8, 1966 The compounds of the present invention where A is CH --CH can be produced from the compound:

on E

o-o-orrornn hereinafter referred to as the fermentation estrogenic substance (F.E.S.) by treatment with an amine or ammonia in the presence of hydrogen anda catalyst, for instance, a Raney nickel catalyst.

The nomenclature used herein is based upon the PBS. compound. For example, the compound of this invention Where A is -CH CH and each R, R and R is hydrogen is referred to as dihydro F.E.S. primary amine. Compounds of this invention where both Rs are alkyl are referred to as dialkyl-, and Where one of the Rs is alkyl as monoalkyl-, dihydro RES. amine.

Treatment of F.E.S. to produce the amine where A is -CH CH is preferably carried out in the presence of an amine with the PBS. suspended or dissolved in a suitable solvent, e.g. an alcohol, preferably a lower alkanol such as methanol, ethanol, and acid, e.g. acetic acid, etc. The ketone and olefinic groups are simultaneously reduced. 'In general, the treatment can be accomplished at ambient temperatures and ambient pressures. Generally temperatures are from about 15 to 150 C. and pressures are from about 1 to atmospheres. In general, from about 0.1 to 5 grams of a Group VIII metal catalyst such as Raney nickel supported on charcoal or kieselguhr are used per gram of F.E.S. An amine having the formula NHR R wherein R and R have the above meaning is present during the reduction. Typical of such compounds are aniline, bromoaniline, ethylamine, methylamine, benzylamine, allylamine, dimethylamine, etc. When R in the above formula is hydrogen, ammonia is present. The amine is present in approximately stoichiometric amounts with an excess of amine being preferable.

The production of compounds of the present invention where A is CH -CH can also be effected by oximation of the PBS. ketone group followed by the simultaneous reduction of the oxime and olefinic groups. The oximation can be accomplished by reaction of a F.E.S. compound such as dimethyl F.E.S., for instance, with hydroxylammonium chloride or a hydroxylammonium chloride reagent solution. The reduction of the oxime and olefinic groups to the primary amine can also be ac complished with hydrogen in the presence of Raney nickel.

In producing compounds of the present invention where R is alkyl, conventional alkylation procedures can be used to replace the H atom of one or both of the OH groups on the benzene ring of F.E.S. with the alkyl group, Alkylated RES. amine compounds can be produced, for example, by first alkylating F.E.S. and then reducing the ketone in the presence of an amine as set forth supra. The alkylation can be by reaction with the corresponding dialkyl sulfates, e.g. dimethyl sulfate, diethyl sulfate, etc. to produce the dialkyl F.E.S. or a monoalkyl F.E.S. with the alkyl group replacing the hydrogen of the hydroxyl group on the benzene ring ortho to the ester group. Furthermore, a monomethyl F.E.S. compound with the methyl group replacing the hydrogen of the hydroxyl group para to the ester .group can be selectively produced using diazomethane.

The following examples are offered to illustrate this invention; however, the invention is not limited to the specific materials, amounts, and precedures set forth. The first example illustrates preparation of a suitable inoculum containing the organism Gibberella zeae (Gordon) NRRL-2830.

Example I A spore sand culture containing Gibberella zeae (Gordon) NRRL2830 was aseptically placed in a sterile tube containing milliliters of Czapek-Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at approximately 25 C. At the end of the incubation period, the medium was washed with 5 milliliters of sterile deionized water and transferred to a sterile tube containing 45 milliliters of CZapek-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25 C. after which the material was available for use in inoculation of a fermentation medium.

The following example illustrates the ferementation of the organism Gibberella zeae (Gordon) NRRL-2830 to produce F.E.S.

Example II To a 2 liter flask were added 300 grams of finely divided corn. The flask and its contents were then sterilized and after sterilization 150 milliliters of sterile deionized water were added. To the mixture in the flask were then added 45 milliliters of the inoculum prepared by the process of Example I and the material was thoroughly mixed. The mixed material Was then incubated for about days at C. in a dark room in a water-saturated atmosphere.

The following example illustrates the recovery of F.E.S. from the fermentation medium.

Example III A 300 gram portion of fermented material produced by the method of Example II was placed in 500 milliliters of deionized water and slurred. The slurry was then heated for about 15 minutes at 75 C., 300 grams of filter aid were then added and the material was filtered. The solid filtered material containing the anabolic substance was then air dried, and 333 grams of the dried cake were then extracted with 500 milliliters of ethanol. This procedure was repeated three more times. The ethanol extract was evaporated to dryness under vacuum to give 6.84 grams of solid material. This solid material was then dissolved in 20 milliliters of chloroform and extracted with milliliters of an aqueous solution containing 5% by weight of sodium carbonate having an adjusted pH of about 11.2. The extraction process was repeated seven more times. The pH of the sodium carbonate extract was then adjusted to 6.2 with hydrochloric acid, to yield an anabolic substance-containing precipitate. The precipitate and the aqueous sodium carbonate extract were then each in turn extracted with 75 milliliters of ethyl ether. This procedure was repeated three more times to yield a light yellow ethereal solution, which was then evaporated to yield 116 milligrams of solid anabolic substance. This material was then subjected to multiple transfer countercurrent distribution using 100 tubes and a solvent system consisting of two parts chloroform and two parts carbon tetrachloride as the lower phase and four parts methanol and one part water as the upper phase, all parts by volume. The solid material obtained from the multiple transfer countercurrent distribution was F.E.S.

The following example illustrates the production of dihydrodimethyl F.E.S. amine.

Example IV F.E.S. in acetic acid was catalytically hydrogenated at room temperature in the presence of PdO at a hydrogen pressure of about p.s.i. to produce dihydro F.E.S. The resulting dihydro F.E.S. was dissolved in a mixture of 10% NaOH and water, and while stirring the solution, dimethyl sulfate was added. The dihydrodimethyl F.E.S. so produced was then oximated as follows: To a solution of 2.65 grams of dihydrodimethyl F.E.S. in a mixture of 50 milliliters ethanol and 40 milliliters pyridine was added 3.5 grams hydroxylammonium chloride. The clear solution was refluxed for 2 hours and evaporated to a volume of 510 milliliters. Water (25 milliliters) was added and the aqueous mixture was extracted with benzene. The dried extract was evaporated to dryness to yield 2.99 grams of a tacky white solid. Crystallization of this product from a mixture of 10 milliliters water and 15 milliliters ethanol provided 1.2 grams of material which melted at 126-l28 C. Recrystallization of 65 milligrams of this material from aqueous ethanol provided 43 milligrams of dihydrodimethyl F.E.S. oxime having a melting point of 130132 C. and analyzing:

Cale. Found (CzoHzgNOs) Percent C (i6. 10 G6. 52 8. 04 8. 03 Percent N 3. 3. 91

The dihydrodimethyl F.E.S. oxime is reduced using a Raney nickel catalyst and 50 p.s.i. of hydrogen over a three hour period to produce dihydrodimethyl F.E.S. amine.

Example V Dihydro F.E.S. amine is produced according to the procedure of Example IV by omitting the alkylation step.

Example VI Dihydrodiethyl F.E.S. amine is produced following the procedure of Example 1V substituting diethyl sulfate for the dimethyl sulfate.

Example VII F.E.S. is reduced to the dihydro F.E.S. primary amine by treatment with ammonia in the presence of hydrogen and Raney nickel catalyst.

Example VIII F.E.S. is reduced to the dihydro F.E.S. N-methylamine by treatment with methylamine in the presence of hydrogen and Raney nickel catalyst.

Example IX F.E.S. is reduced to the dihydro F.E.S. N-phenylamine by treatment with aniline in the presence of hydrogen and Raney nickel catalyst.

The following example illustrates the oximation of F.E.S. and the reduction of the oxime and olefinic groups to produce dihydro F.E.S. amine.

Example X where (2 grams of F.E.S. liberate an amount of acid corresponding to b milliliters base of normality n. This procedure when applied to a total of 53 milligrams Calc. Found 1a 2a05N) Percent C H 64. 84 63. 62 Percent H 6. 95 7.05 Percent N 4. 20 4. 15

B. The F.E.S. oxime is reduced to dihydro F.E.S. amine by reducing the oxime and olefinic groups using Raney nickel catalyst and 50 p.s.i. of hydrogen over a three hour period.

The following example illustrates the production of monomethyland dimethyhdihydro F.E.S. amine, the monomethyl F.E.S. amine having a methyl group which replaced the hydrogen of the hydroxyl group on the henzene ring ortho to the ester group.

Example XI Dimethyl sulfate (5 milliliters) was added to a solution of 2.24 grams RES. in 80 milliliters of a NaOH solution and milliliters of Water. The mixture was stirred for one-half hour at 1820 C. (cooling bath) and an additional 5 milliliters of dimethyl sulfate was added. After an additional 70 minutes of stirring at 20-26 C., the solid precipitate, solid A, was collected by filtration, washed with water and dried. The filtrate from solid A was acidified with milliliters 12 N H 80 to yield a second precipitate, solid B, which was collected, washed with water, and dried.

Solid A (0.79 gram having a melting point of 114- 118 C.) was recrystallized from a mixture of 10 milliliters water and 15 milliliters ethanol to yield 0.66 gram dimethyl F.E.S. having a melting point of 1081l.0 C.

Solid B (1.39 grams having a melting point of 152- 162 C.) was recrystallized twice from a mixture of water and alcohol to yield 0.80 gram of monomethyl F.E.S. product having a melting point of 169174 C. Analysis of solid B showed:

The ketone group of each of the dimethyl F.E.S. and monomethyl F.E.S. are oximated and the oxime and olefinic groups of each are reduced according to the procedure of Example X.

Example XII Monomethyl dihydro F.E.S. with the methyl group replacing the hydrogen of the hydroxyl group on the benzene ring para to the ester group was prepared by the following procedure.

Nitrosomethylurea in an amount of 1.2 grams was slowly added to a cold mixture of 3.6 milliliters of 50% potassium hydroxide and 17 milliliters of ether. After a few minutes the yellow ether layer of the mixture was decanted, dried over potassium hydroxide, and then added to a solution of 0.30 gram F .E.S. in 17 milliliters of ether. The resulting yellow mixture was left overnight in a loosely stoppered flask .and then ether and diazomethane were evaporated off using a steam bath. The remaining gummy residue was crystallized by adding 3 milliliters of water, heating to 60 C., and adding ethanol almost to solution. On cooling, crystals formed, yielding 0.137 gram of product having a melting point of 111-116 C. which was recrystallized in the same way to yield 0.082 gram of monomethyl F.E.S. having a melting point of 120122 C. and the following analysis:

Cale. Found (CIQH2405) Percent; C 68. 7 68.3 Percent H 7. 28 7.38 Percent OCH; 9.38 9.17

The ketone group of the monomethyl F.E.S. is oximated and the oxime and olefinic groups are reduced according to the procedure of Example X.

Example XIII The compound:

CHaO- is produced by reaction of dimethyl dihydro PBS. and methylamine in the presence of hydrogen and Raney nickel catalyst.

Example XIV The compound:

(HI-Q (cHa/ is produced according to Example XIII substituting metatoluidine for methylamine.

Example XV Six head of cattle are fed a daily ration including a mixture of alfalfa hay and ground corn cobs containing from 1 to 20 ounces of dihydro F.E.S. primary amine per hundred pounds of ration.

Example XVI wherein A is -CH CH R is selected from the group consisting of hydrogen and lower alkyl, R is selected from the group consisting of hydrogen, lower alkyl and aryl and R is hydrogen.

2. The compound of claim 1 wherein each R and R is hydrogen.

3. The compound of claim 1 wherein each R is lower alkyl and R is hydrogen.

4. The compound of claim 1 wherein each R is hydrogen and R is lower alkyl.

5. The compound of claim 1 wherein each R is hydrogen and R is phenyl'.

6. The compound of claim 1 wherein each R is lower alkyl and R is lower alkyl.

7. An animal feed comprising a nutritional diluent and growth promoting amounts of the compound of claim 1.

8. The compound of claim 1 wherein R is methyl and R is hydrogen.

9. The compound of claim 1 wherein R is ethyl and R is hydrogen.

10. The compound of claim 1 wherein R is hydrogen and R is methyl.

11. The compound of claim 1 wherein the R ortho to the ester group is methyl and the other R is hydrogen and R is hydrogen.

12. The compound of claim 1 wherein the R para to the ester group is methyl and the other R is hydrogen and R is hydrogen. 7 p

13. The compound of claim 1 wherein R is methyl 'and R is methyl.

14. The compound of claim 1 wherein R is hydrogen and R is m-tolyl.

15. An animal feed comprising a nutritional diluent and growth promoting amounts of the compound of claim 2.

16. An animal feed comprising a nutritional diluent and growth promoting amounts of the compound of claim 3.

17. An animal feed comprising a nutritional diluent and growth promoting amounts of the compound of claim 4.

18. An animal feed comprising a nutritional diluent and growth promoting amounts of the compound of claim 5.

19. An animal feed comprising a nutritional diluent and growth promoting amounts of claim 6.

20. An animal feed comprising a and growth promoting amounts of claim 8.

21. An animal feed comprising a and growth promoting amounts of claim 9.

22. An animal feed comprising a and growth promoting amounts of claim 10.

23. An animal feed comprising a and growth promoting amounts of claim 11.

24. An animal feed comprising a and growth promoting amounts of claim 12.

25. An animal feed comprising a and growth promoting amounts of claim 13. v

26. An animal feed comprising a and growth promoting amounts of the compound of nutritional diluent the compound of nutritional diluent the compound of nutritional diluent the compound of nutritional diluent the compound of nutritional diluent the compound of nutritional diluent the compound of nutritional diluent the compound of claim 14.

References Cited by the Examiner UNITED STATES PATENTS 2,842,051 8/1958 Brian et al. 99-2 OTHER REFERENCES Stob et al.: Nature, vol. 196, page 1318, Dec. 29, 1962.

A. LOUIS MONACELL, Primary Examiner. 

7. AN ANIMAL FEED COMPRISING A NUTRITIONAL DILUENT AND GROWTH PROMOTING AMOUNTS OF THE COMPOUND OF CLAIM
 1. 